Novel human PD-L1 protein antibody

Abstract

The invention discloses a novel human PD-L1 protein antibody, belongs to the technical field of biological medicines, and relates to the novel human PD-L1 protein antibody which is designed through methods such as butt joint and dynamic simulation. The invention discloses an expression and purification technology of the novel antibody, and a novel antibody protein with the purity of more than 90% is successfully obtained. The biological preparation has very high application value, and a new antibody preparation is provided for treating related diseases taking PD-L1 as target protein.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to a novel antibody against human PD-L1 protein. Background Art

[0002] PD-1 (Programmed Death 1) is a type I transmembrane glycoprotein belonging to the CD28 family, while PD-L1 (Programmed Death-Ligand 1) is a type I transmembrane glycoprotein with a size of 40KDa that exists as programmed death receptor-ligand 1.

[0003] In the human body, PD-L1 protein is continuously expressed in some specific tissues, including the liver, lungs, tonsils, and immune-related tissues such as eyes and placenta. In addition, human PD-L1 is also present in T cells, B cells, monocytes, dendritic cells, and macrophages. In the human body, the binding of PD-L1 to PD-1 leads to T cell apoptosis, thereby reducing the immune response. This negative regulatory mechanism plays a key role in tumor immunity. In 2004, researchers first observed the expression of PD-L1 in tissue samples from patients with non-small cell lung cancer. Since then, it has been found that human PD-L1 is expressed in tissues of patients with various cancers, including but not limited to gastric cancer, lung cancer, liver cancer, and colon cancer. Therefore, although the immune system has mechanisms to monitor and clear these antigens, some tumor cells can still successfully escape the control of immune attacks under the regulation of PD-L1. In addition, a variety of chronic and acute viruses also use PD-L1 signals to evade human immune surveillance. A study showed that the expression of PD-L1 on bone marrow-derived dendritic cells of HIV-infected patients increased significantly, and after antiviral treatment, the expression of PD-L1 was negatively correlated with the number of T cells. In addition, studies have found that the expression of PD-L1 on T lymphocytes and dendritic cells of chronic HBV-infected patients also increased. When infection occurs, it causes infected cells to express high levels of PD-L1 and induces CD8+T cells to express PD-1, thereby inhibiting T cell function and causing the loss of effector T cells.

[0004] In molecular biology research and the biotechnology industry, the expression and purification of target proteins in prokaryotic systems are basic tools that help to deeply explore the functions and properties of proteins and promote the vigorous development of modern biology. Usually, the expression and purification of target proteins go through the following steps: the target gene is cleverly cloned into a suitable vector, introduced into a prokaryotic expression system, the protein is efficiently expressed under induction conditions, and the target protein is accurately separated and purified by means of fusion tags and chromatography columns. This application uses this technology to quickly obtain and separate a new antibody against human PD-L1. Summary of the invention

[0005] 1. Technical problems to be solved: In view of the problems existing in the prior art, the purpose of the present invention is to provide a new antibody of human PD-L1 protein to block the binding between PD-1 and PD-L1, thereby enhancing T cell function to achieve the function of promoting the immune system to kill tumor cells.

[0006] 2. Technical solution: To solve the above problems, the present invention adopts the following technical solutions.

[0007] A novel human PD-L1 protein antibody, including the steps of designing and analyzing novel artificial antibody sequences at the computer level: S1. Molecular docking technology was used to simulate the interaction between antibodies and PD-L1 and predict possible binding modes; S2. Design the amino acid sequence of the antibody using the ProteinMPNN algorithm, which generates amino acid sequences with specific functions based on the three-dimensional structural information of proteins; S3. The designed antibody sequence is evaluated for binding affinity using the Prodigy platform to ensure that it has high affinity to human PD-L1.

[0008] A further improvement is that: the method for preparing the protein also includes: S4, constructing and culturing the expression strain of the tagged recombinant protein; S5, cleavage and purification of the protein, Ni-NTA affinity chromatography and gel filtration chromatography; S6. Determination of affinity for receptor protein.

[0009] A further improvement is that the protein expression method in step S4 comprises the following steps: S41. Fully synthesize the designed plasmid containing the nucleic acid sequence of the novel human PD-L1 antibody, and perform restriction digestion and ligation using NcoI and XhoI restriction enzymes to ensure that the sequence is correctly inserted into the pET-28a expression vector; S42, adding the synthesized expression plasmid to BL21 (DE3) competent cells and placing them on ice for 30 minutes to reduce the damage of heat shock to the cells; S43, heat shock treatment at 42°C for 50 seconds to promote the binding of the plasmid to the host cells; S44. Quickly cool the cells on ice for 2 minutes to reduce the continued effects of heat shock on the cells. Then, add 100 μL of LB liquid medium to the cells and incubate at 37°C for 45 minutes to promote cell recovery and growth; S45, spreading the revived cells on LB solid medium containing Kan resistance, and culturing them in a 37°C constant temperature incubator for 12 hours to screen out successfully transformed cells; S46, pick a single colony and transfer it to 30 mL of LB liquid medium containing Kan resistance, and culture it on a shaker at 37°C and 200 rpm for 10 hours to obtain a sufficient number of cells; S47, transfer the culture to 1 L LB liquid medium containing Kan resistance, and continue to culture on a shaker at 37°C and 200 rpm until the OD600 reaches 0.8, indicating that the cell density has reached the appropriate expression condition; S48. Add 0.5 mM IPTG and induce expression for 6 hours at 30°C and 200 rpm on a shaker to promote the expression of the target protein.

[0010] A further improvement is that the purification method of protein cleavage purification, Ni-NTA affinity chromatography and gel filtration chromatography in step S5 comprises the following steps: S51, collect the cultured strain containing the recombinant antibody by centrifugation at 5000 rpm for 10 minutes; resuspend the bacteria in 30 mL of PBS buffer, disrupt the cells by ultrasonic disruption, and then centrifuge at 13000 rpm for 30 minutes at 4°C to collect the supernatant containing the recombinant protein; S52, the supernatant was added to the pre-cooled Ni-NTA affinity chromatography column and incubated at 4°C for 25 minutes to promote the affinity binding of the recombinant protein to the Ni-NTA resin on the nickel column through its His tag. After the unbound impurities flowed out with the supernatant, the column was washed with 5-6 column volumes of Washing buffer to remove impurities; S53, add 15 mL of Elution buffer and incubate at 4°C for 5 minutes to dissolve and elute the recombinant protein bound to the column; S54, the purified protein was analyzed by SDS-PAGE electrophoresis; S55. Equilibrate the column with General buffer for 2-3 column volumes, filter the Elution buffer sample containing the recombinant protein through a 0.22 μm filter membrane, and load it onto a Superdex200 chromatography column at a flow rate of 0.8 mL / min.

[0011] A further improvement is that the affinity determination in step S6 comprises the following steps: S61, PD-L1 was selected as the receptor protein, and PD-L1 was incubated with Cy5 dye in the dark at 4°C for 30 minutes; S62, free dye was effectively removed by gel filtration chromatography, and finally the stained PD-L1 with a concentration of 200 nM was obtained; S63, the novel antibody was diluted stepwise in 16 steps, with the highest concentration being 50 μM, and stained PD-L1 was added sequentially with a final concentration of 100 nM; S64, after mixing, incubate at 4°C for 30 minutes, centrifuge at 13000 rpm for 10 minutes, and take the supernatant for affinity measurement; S65, MST technology is used to determine affinity by measuring the thermophoretic movement of fluorescently labeled molecules. It does not require fixation and can be measured in biological solutions. It does not require purification and has low sample consumption.

[0012] A further improvement is that the expression strain in step S4 is set to the Escherichia coli strain BL21 (DE3).

[0013] A further improvement is that: the culture medium and reagents in the protein preparation process include LB liquid culture medium, LB solid culture medium, and Ni-NTA affinity chromatography buffer; The LB liquid culture medium includes 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride. The LB solid medium includes 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar; The Ni-NTA affinity chromatography buffer and gel filtration buffer solution include Washing buffer, Elution buffer and General buffer, wherein; The washing buffer includes 2.7 mM potassium chloride, 137 mM sodium chloride, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate, and 30 mM imidazole; The elution buffer includes 2.7 mM potassium chloride, 137 mM sodium chloride, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate, and 250 mM imidazole; The General buffer includes 2.7 mM potassium chloride, 137 mM sodium chloride, 10 mM disodium hydrogen phosphate, and 1.8 mM potassium dihydrogen phosphate.

[0014] 3. Beneficial effects: Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects: The present invention is reasonably designed, and a novel antibody for PD-L1 protein is designed by docking, dynamic simulation and other methods. The present invention discloses the expression and purification technology of the novel antibody, and successfully obtains a novel antibody protein with a purity of more than 90%; so as to realize that the biological preparation has a high application value, and provides a new antibody preparation for the treatment of related diseases with PD-L1 as the target protein; The present invention constructs a tag recombinant protein for expressing and purifying a small peptide, wherein the tag recombinant protein includes a purification tag and a target protein. The purification tag uses a 6*His-tag. The 6*His-tag (HHHHHH) is a short peptide composed of 6 histidines. Preferably, the 6*His-tag is used to significantly improve the purification efficiency; The present invention provides an isolated nucleic acid molecule encoding an antibody or a functional fragment thereof that can bind to human PD-L1, and an expression vector and a host cell comprising the nucleic acid molecule or a complementary sequence thereof; The present invention prepares a novel antibody that binds to PD-L1 by affinity chromatography, gel filtration chromatography and the like; The present invention analyzes the AlphaFold structure of a novel antibody that binds to PD-L1; The present invention provides a method for inhibiting the binding of PD-L1 protein and PD-1 protein by using a novel artificial antibody; The present invention measures the affinity of the novel artificial antibody to PD-L1 by using microcalorimetry technology (MST).

[0015] It should be noted that the structures not introduced in the present invention are the same as the prior art or can be implemented by using the prior art, and are not described in detail here because they do not involve the design points and improvement directions of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the predicted structural analysis diagram of the protein AlphaFold of the present invention; Figure 2 The key amino acid sites for the interaction between the novel PD-L1 antibody (gray) of the present invention and human PD-L1 (black); Figure 3 is the SDS-PAGE diagram of the protein expression of the present invention; Figure 4 This is a gel filtration diagram of the protein purification of the present invention; Figure 5 This is the result of MST determination of the antibody of the present invention and the receptor protein. DETAILED DESCRIPTION

[0017] To facilitate understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Several embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0018] The present invention will be described in more detail below through specific implementation cases and accompanying diagrams. It should be noted that the implementation cases described are only used to illustrate the present invention and do not impose any restrictions on it. The plasmid vectors, materials and reagents used in the implementation cases, unless otherwise specified, can be obtained through commercial channels. Experimental methods that do not specify the conditions are usually carried out under standard conditions or follow the recommended conditions provided by the manufacturer. The Escherichia coli strain BL21 (DE3) used in the experiment was purchased from Sangon Biotech (Shanghai) Co., Ltd., and it can grow normally in conventional LB medium. In order to promote its growth, we chose 50μg / mL Kan as the antibiotic concentration. For vector cloning and protein expression purification steps, please refer to the Molecular Cloning Experiment Guide.

[0019] The media and reagents used include: LB liquid medium: including 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride, the volume was fixed to 1 L, and then sterilized under high pressure at 121° C. for 20 minutes.

[0020] LB solid culture medium: including 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, and 15g of agar. The volume was adjusted to 1L, and then sterilized under high pressure at 121°C for 20 minutes. After cooling slightly to about 60°C, it was poured into the clean bench to prepare the plate.

[0021] Ni-NTA affinity chromatography buffer: Washing buffer: 2.7 mM KCl, 137 mM NaCl, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate, 30 mM imidazole.

[0022] Elution buffer: 2.7 mM KCl, 137 mM NaCl, 10 mM NaHPO4, 1.8 mM KH2PO4, 250 mM Imidazole.

[0023] Gel filtration chromatography buffer: General buffer: 2.7 mM potassium chloride, 137 mM sodium chloride, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate. After filtration, degassing was performed by ultrasonication at 40% power for 30 minutes.

[0024] 1. Design and analysis of new artificial antibody sequences at the computer level: The present invention provides a scheme for developing a new artificial antibody using a computer-aided design method, which can specifically bind to the human PD-L1 protein for use in cancer immunotherapy. In the design process, molecular docking technology is first used to simulate the interaction between the antibody and PD-L1 and predict possible binding modes. Subsequently, the amino acid sequence of the antibody is designed using the ProteinMPNN algorithm, which generates an amino acid sequence with specific functions based on the three-dimensional structural information of the protein. Finally, the designed antibody sequence is evaluated for binding strength using the Prodigy platform to ensure that it has high affinity with human PD-L1.

[0025] AlphaFold was used to predict the structure and retrograde behavior of the novel antibody, and protein visualization software such as pymol was used to analyze the structure of the designed novel antibody. It was found that the main structure of the novel antibody of human PD-L1 is mainly composed of three helices, such as Figure 1 After analyzing the structure, it was found that in the binding process, there are 6 amino acids (D4, Q8, T12, Y34, K38, Y39) as key amino acids involved in the binding of the new antibody to human PD-L1 and human PD-L1, such as Figure 2 .

[0026] As an antibody to the PD-L1 protein, this protein can be used to block the binding between PD-L1 and PD-1, thereby enhancing the immune system's killing of tumor cells and ultimately achieving the purpose of disease treatment.

[0027] 2. Protein Preparation 1) Construct and culture the expression strain of the tagged recombinant protein: The present invention relates to a method for expressing a novel human PD-L1 antibody, which comprises the following steps: first, a designed plasmid containing a nucleic acid sequence of a novel human PD-L1 antibody is fully synthesized at Sangon Biotech (Shanghai) Co., Ltd., and NcoI and XhoI restriction enzymes are used for enzyme cutting and ligation to ensure that the sequence is correctly inserted into the pET-28a expression vector. Subsequently, the synthesized expression plasmid is added to BL21 (DE3) competent cells and left on ice for 30 minutes to reduce the damage of heat shock to the cells. Next, a heat shock treatment is performed for 50 seconds at 42°C to promote the binding of the plasmid to the host cells, and then the cells are quickly placed on ice for 2 minutes to reduce the continuous effect of heat shock on the cells. Then, 100 μL of LB liquid culture medium is added to the cells and incubated at 37°C for 45 minutes to promote cell recovery and growth. Next, the revived cells are spread on a solid LB culture medium containing Kan resistance and cultured in a constant temperature incubator at 37°C for 12 hours to screen out successfully transformed cells. After that, single colonies were selected and transferred to 30 mL of LB liquid medium containing Kan resistance, and cultured on a shaker at 37°C and 200 rpm for 10 hours to obtain a sufficient number of cells. Subsequently, the culture was transferred to 1 L of LB liquid medium containing Kan resistance and continued to be cultured on a shaker at 37°C and 200 rpm until OD600 reached 0.8, indicating that the cell density reached the appropriate expression conditions. Finally, 0.5 mM IPTG was added on a shaker at 30°C and 200 rpm to induce expression for 6 hours to promote the expression of the target protein.

[0028] 2) Protein purification: In the present invention, we describe in detail a method for efficiently purifying a novel human PD-L1 antibody, which includes three main steps: lysis purification, Ni-NTA affinity chromatography and gel filtration chromatography.

[0029] First, the cultured strain containing the recombinant antibody was centrifuged at 5000 rpm for 10 minutes to collect the cells. The cells were resuspended in 30 mL of PBS buffer, and then ultrasonically disrupted, and then centrifuged at 13000 rpm for 30 minutes at 4°C to collect the supernatant containing the recombinant protein.

[0030] Next, the supernatant was added to the pre-cooled Ni-NTA affinity chromatography column and incubated at 4°C for 25 minutes to promote the affinity binding of the recombinant protein to the Ni-NTA resin on the nickel column through its His tag. After the unbound impurities flowed out with the supernatant, the chromatography column was washed with 5-6 column volumes of Washing buffer to remove the impurities. Finally, 15 mL of Elution buffer was added and incubated at 4°C for 5 minutes to dissolve and elute the recombinant protein bound to the column. The purified protein was analyzed by SDS-PAGE electrophoresis, as shown in Figure 2. Figure 3 shown.

[0031] In order to further improve the purity of the protein, we used AKTA Pure instrument for gel filtration chromatography. Superdex200 purification column and General buffer were used in the experiment. Before the experiment, the column was equilibrated with General buffer for 2-3 column volumes. Then, the Elution buffer sample containing the recombinant protein was filtered through a 0.22μm filter membrane and loaded onto the Superdex200 chromatography column at a flow rate of 0.8 mL / min. Based on the peak position appearing in the UV absorption peak graph, we determined Figure 4 The second peak in the figure is the purification peak of the prepared antibody protein, indicating that the highly pure tagged recombinant protein was successfully purified.

[0032] 3. Affinity determination: In order to verify the affinity between the new antibody and the target protein, this study used microthermophoresis (MST) technology for measurement. Microthermophoresis is an optical method for characterizing the properties of biomolecules. It obtains information such as the mode and kinetic constants of molecular interactions by detecting the changes in the electrophoretic mobility of biomolecules in a temperature gradient.

[0033] In the experiment, we selected PD-L1 as the receptor protein and incubated PD-L1 with Cy5 dye in the dark for 30 minutes at 4°C. Cy5 is a commonly used fluorescent dye with a high molar extinction coefficient that can produce bright fluorescent signals and is suitable for fluorescent labeling of biological molecules. The free dye was effectively removed by gel filtration chromatography, and finally the stained PD-L1 with a concentration of 200 nM was obtained. Gel filtration chromatography is a separation technique based on molecular size that can effectively separate dye-bound proteins from unbound dye.

[0034] Subsequently, the new antibody was diluted stepwise in 16 gradients, with the highest concentration being 50 μM, and stained PD-L1 with a final concentration of 100 nM was added in sequence. After mixing, incubate at 4°C for 30 minutes, centrifuge at 13,000 rpm for 10 minutes, and take the supernatant for affinity measurement. MST technology determines affinity by measuring the thermophoretic movement of fluorescently labeled molecules. It does not require fixation, is measured in biological solutions, does not require purification, and has low sample consumption.

[0035] The measurement results are as follows Figure 5 The measured KD value is 3.70E-07 M, proving that the designed new antibody does have a certain affinity with PD-L1. The KD value, or dissociation constant, is an important parameter that describes the affinity between molecules. The smaller the value, the stronger the affinity.

[0036] The novel humanized PD-L1 antibody designed in this application has the following amino acid sequence in its protein: MGHHHHHHGLADTVAQATATAEAYVKAGDKERADKVLEFLEYLAKKYNNPAIAKKVAEIRALME The nucleotide sequence of the protein is as follows: ATGGGCCATCATCATCATCATCATGGCCTGGCGGATACCGTGGCGCAGGCGACCGCGACC GCGGAAGCGTATGTGAAAGCGGGCGATAAAGAACGCGCGGATAAAGTGCTGGAATTTCTG GAATATCTGGCGAAAAAATATAACAACCCGGCGATTGCGAAAAAAGTGGCGGAAATTCGC GCGCTGATGGAA In summary, this application provides a new antibody to human PD-L1 protein, and successfully induced expression in the E. coli system to achieve rapid production of the antibody. The exact interaction between the new antibody and the receptor protein PD-L1 was verified by microscale thermophoresis (MST) technology, laying the foundation for the further application of the antibody. The technical scope of this invention is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

[0037] The above-described embodiments only express a certain implementation mode of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A novel human PD-L1 protein antibody, characterized in that: Including the design and analysis steps of new artificial antibody sequences at the computer level: S1. Molecular docking technology was used to simulate the interaction between antibodies and PD-L1 and predict possible binding modes; S2. Design the amino acid sequence of the antibody using the ProteinMPNN algorithm, which generates amino acid sequences with specific functions based on the three-dimensional structural information of proteins; S3. The designed antibody sequence is evaluated for binding affinity using the Prodigy platform to ensure that it has high affinity to human PD-L1.

2. A novel human PD-L1 protein antibody according to claim 1, characterized in that: Also included is a method for preparing the protein: S4, constructing and culturing the expression strain of the tagged recombinant protein; S5, cleavage and purification of the protein, Ni-NTA affinity chromatography and gel filtration chromatography; S6. Determination of affinity for receptor protein.

3. A novel human PD-L1 protein antibody according to claim 2, characterized in that: The protein expression method in step S4 comprises the following steps: S41. Fully synthesize the designed plasmid containing the nucleic acid sequence of the novel human PD-L1 antibody, and perform restriction digestion and ligation using NcoI and XhoI restriction enzymes to ensure that the sequence is correctly inserted into the pET-28a expression vector; S42, adding the synthesized expression plasmid to BL21 (DE3) competent cells and placing them on ice for 30 minutes to reduce the damage of heat shock to the cells; S43, heat shock treatment at 42°C for 50 seconds to promote the binding of the plasmid to the host cells; S44. Quickly cool the cells on ice for 2 minutes to reduce the continued effects of heat shock on the cells. Then, add 100 μL of LB liquid medium to the cells and incubate at 37°C for 45 minutes to promote cell recovery and growth; S45, spreading the revived cells on LB solid medium containing Kan resistance, and culturing them in a 37°C constant temperature incubator for 12 hours to screen out successfully transformed cells; S46, pick a single colony and transfer it to 30 mL of LB liquid medium containing Kan resistance, and culture it on a shaker at 37°C and 200 rpm for 10 hours to obtain a sufficient number of cells; S47, transfer the culture to 1 L LB liquid medium containing Kan resistance, and continue to culture on a shaker at 37°C and 200 rpm until the OD600 reaches 0.8, indicating that the cell density has reached the appropriate expression condition; S48. Add 0.5 mM IPTG and induce expression for 6 hours at 30°C and 200 rpm on a shaker to promote the expression of the target protein.

4. A novel human PD-L1 protein antibody according to claim 2, characterized in that: The purification method of protein cleavage purification, Ni-NTA affinity chromatography and gel filtration chromatography in step S5 comprises the following steps: S51, collect the cultured strain containing the recombinant antibody by centrifugation at 5000 rpm for 10 minutes; resuspend the bacteria in 30 mL of PBS buffer, disrupt the cells by ultrasonic disruption, and then centrifuge at 13000 rpm for 30 minutes at 4°C to collect the supernatant containing the recombinant protein; S52, the supernatant was added to the pre-cooled Ni-NTA affinity chromatography column and incubated at 4°C for 25 minutes to promote the affinity binding of the recombinant protein to the Ni-NTA resin on the nickel column through its His tag. After the unbound impurities flowed out with the supernatant, the column was washed with 5-6 column volumes of Washing buffer to remove impurities; S53, add 15 mL of Elution buffer and incubate at 4°C for 5 minutes to dissolve and elute the recombinant protein bound to the column; S54, the purified protein was analyzed by SDS-PAGE electrophoresis; S55. Equilibrate the column with General buffer for 2-3 column volumes, filter the Elution buffer sample containing the recombinant protein through a 0.22 μm filter membrane, and load it onto a Superdex200 chromatography column at a flow rate of 0.8 mL / min.

5. A novel human PD-L1 protein antibody according to claim 2, characterized in that: The affinity determination in step S6 comprises the following steps: S61, PD-L1 was selected as the receptor protein, and PD-L1 was incubated with Cy5 dye in the dark at 4°C for 30 minutes; S62, free dye was effectively removed by gel filtration chromatography, and finally the stained PD-L1 with a concentration of 200 nM was obtained; S63, the novel antibody was diluted stepwise in 16 steps, with the highest concentration being 50 μM, and stained PD-L1 was added sequentially with a final concentration of 100 nM; S64, after mixing, incubate at 4°C for 30 minutes, centrifuge at 13000 rpm for 10 minutes, and take the supernatant for affinity measurement; S65, MST technology is used to determine affinity by measuring the thermophoretic movement of fluorescently labeled molecules. It does not require fixation and can be measured in biological solutions. It does not require purification and has low sample consumption.

6. A novel human PD-L1 protein antibody according to claim 2, characterized in that: The expression strain in step S4 is set to the Escherichia coli strain BL21 (DE3).

7. A novel human PD-L1 protein antibody according to claim 2, characterized in that: The culture medium and reagents used in the protein preparation process include LB liquid culture medium, LB solid culture medium, and Ni-NTA affinity chromatography buffer; The LB liquid culture medium includes 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride. The LB solid medium includes 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar; The Ni-NTA affinity chromatography buffer and gel filtration buffer solution include Washing buffer, Elution buffer and General buffer, wherein; The washing buffer includes 2.7 mM potassium chloride, 137 mM sodium chloride, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate, and 30 mM imidazole; The elution buffer includes 2.7 mM potassium chloride, 137 mM sodium chloride, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate, and 250 mM imidazole; The General buffer includes 2.7 mM potassium chloride, 137 mM sodium chloride, 10 mM disodium hydrogen phosphate, and 1.8 mM potassium dihydrogen phosphate.

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